Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host, where the bacterium down modulates the macrophage immune functions, a phenomenon similar to tumor-infiltrated macrophage phenotypes in cancer. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. Using models of either primary macrophage or cancer cell line infection with F. tularensis, we have expanded our knowledge of the intracellular cycle of this pathogen, by showing that phagosomal escape occurs rapidly after phagocytosis (Checroun et al., 2006, PNAS, 103:14578) and that the efficiency of phagosomal disruption depends upon the conditions encountered within the early Francisella-containing phagosome (FCP). Upon acidification, the early FCP provides environmental cues for the induction of the FPI genes, which encode a Type 6 secretion system required for phagosomal escape (Chong et al., 2008, Infect. Immun., 76:5488). To further investigate the role of the FPI in intracellular pathogenesis, we have an ongoing collaboration with Dr Karl Klose (University of Texas at San Antonio) in which we have demonstrated the functionality of the FPI Type 6 secretion system. A manuscript detailing this work is under revision. We have also examined the contribution of acid phosphatases (Acp) in phagosomal escape of virulent strains, as these proteins have been shown to play such a role in the low virulence subspecies novicida. Using genomic comparisons and systematic deletions of acp genes, we have discovered that most acp genes have been disrupted through genome reduction in virulent strains and the most conserved genes between Francisella subspecies do not play any role in pathogenesis of virulent strains. These findings, which are part of a submitted manuscript, highlight functional differences in the pathogenesis of Francisella subspecies. Another line of ongoing investigation deals with understanding how immune stimuli such as cytokines modulate the intracellular cycle of Francisella. We have established that IFN&#947;activation of macrophages does not affect the ability of Francisella to disrupt its early phagosome, yet restricts cytosolic growth of the bacterium in a manner that is independent of reactive oxygen or nitrogen species or tryptophan depletion. This study is the subject of a manuscript under revision. In our efforts to identify Francisella genes that are important for intracellular pathogenesis, we have worked in collaboration with the RTB/RTS Genomics Unit at RML and established the intracellular transcriptome of the prototypical virulent strain Schu S4 of F. tularensis. In addition to revealing key elements of the intracellular biology of Francisella, this study has identified Francisella-specific genes of unknown functions as upregulated during the intracellular cycle. We have generated deletion mutants of these genes, using a method of allelic replacement we have developed, and identified 3 (FTT0369c, FTT0383 and FTT1676) that were required for intracellular growth in macrophages and virulence in a mouse model of tularemia, developed by Katy Bosio. This study, which demonstrates that novel virulence determinants of Francisella can be identified based on their intracellular expression profiles, was published this year (Wehrly et al., 2009, Cell. Microbiol, 11:1128). Interestingly, mutants in FTT0369c and FTT1676 conferred high levels of protection of mice against a pulmonary challenge with a virulent strain, suggesting that such mutants have some potential as genetically defined, live vaccine strains against tularemia.